Abstract The antigen receptors on lymphocytes play pivotal roles in controlling the balance between tolerance and immunity. In B cells, the B cell antigen receptor (BCR) transmits signals that affect lymphocyte survival, proliferation, and differentiation. The BCR not only affects immune responses by transducing signals - it also internalizes antigen for presentation to the T cells. The relationship of BCR signaling and antigen internalization is controversial. Moreover, the molecular mechanisms by which co-receptors influence these processes are not known. The long-term objectives of the proposed research are to develop a suite of chemical tools to investigate B cell signaling, and to use information gained from them to control B cell signaling and the immune responses that result. The Specific Aims follow. The first aim is to dissect the relationships between antigen signaling, internalization, and trafficking. New advances in polymer chemistry will be used to synthesize multivalent antigens that can illuminate antigen internalization and trafficking in live cells. Using latent fluorophores and modern imaging methods, we shall determine whether the processes of antigen signaling and internalization are linked or disconnected. In addition, studies in this aim will explore new approaches to deliver protein or peptide antigens to B cells for promoting immune responses. The second aim is to generate ligands that can bind to both the negative co-receptor CD22 and the BCR, and to investigate whether these ligands can influence signaling, and antigen internalization, and trafficking. We postulate that antigens that display oligosaccharides that bind CD22 can attenuate BCR signaling, and synthetic probes will be used to test this hypothesis. We anticipate that these studies could reveal an important role for glycosylation in regulating immune responses. In addition, the studies proposed in Aim 2 can yield new strategies to suppress activation of specific B cell populations, thereby promoting tolerance. The third aim is focused on using mass spectrometry to analyze how the synthetic antigens used in Aims 1 and 2 influence protein phosphorylation in B cells. The goal of this aim is to understand the molecular basis for the influence of the synthetic antigens on B cell signaling. We anticipate that the proposed investigations can give rise to new chemical biology strategies for eliciting immunity or tolerance.